Center for Autism Research and Treatment, Semel Institute for Neuroscience and Human Behavior, Department of Psychiatry, University of California, Los Angeles, Los Angeles, CA 90095, USA; Department of Biology, The University of Texas at Tyler, Tyler, TX 75799, USA.

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Department of Cell and Developmental Biology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, PA 19104, USA.

Child Study Center, Yale School of Medicine, New Haven, CT 06510, USA; Program on Neurogenetics, Yale School of Medicine, New Haven, CT 06510, USA; Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA; Department of Psychiatry, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address: matthew.state@ucsf.edu.

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Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA. Electronic address: antonio.giraldez@yale.edu.

Abstract

Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism.

A. Rank-sorting of the anti-correlating dataset with respect to estrogenic compounds shows significant enrichment of estrogenic agents in the top ranks (p=0.0003 by random permutation). Black lines indicate drugs defined as having estrogenic activity (25 compounds in total).B. Hierarchical clustering of the behavioral fingerprints of cntnap2aΔ121/Δ121cntnap2b31i/31i larvae exposed to 14 psychoactive agents at three doses each relative to the wild-type + no drug fingerprint. Each rectangle in the clustergram represents the Z-score of drug-exposed mutants relative to untreated wild-type (magenta, higher than wild-type; cyan, lower than wild-type).C. Magnified sections of the clustergram show relative suppression of the mutant fingerprint by biochanin A (0.1 μM) compared to risperidone (0.001 μM). The red box highlights parameters that measure nighttime activity.D. Pairwise Euclidean distances (“Differential Drug Effects”) between the mutant responses to psychoactive agents compared to untreated wild-type in the PCA (). Biochanin A (0.1 μM) produces the strongest suppression of the mutant phenotype in this assay, with the fewest effects on other behavioral parameters.E, F. Dose-response effects of biochanin A (E) and risperidone (F) on nighttime activity at 5 dpf (p=0.0001, biochanin A; p=0.0034, risperidone, two-way ANOVA, gene x drug interaction). While there is some experimental variability in the baseline activity of both wild-type and mutant larvae, nighttime hyperactivity is consistently observed.G, H. Effect of the blind addition of biochanin A (0.1–1 μM) or DMSO on activity at night (G) and day (H) in the progeny of incrosses of cntnap2aΔ25/+cntnap2bΔ7/+ fish at 5 dpf (*p=0.045, two-way ANOVA, gene x dose interaction).